Sequential and Ordered Assembly of E1 Initiator Complexes on the Papillomavirus Origin of DNA Replication Generates Progressive Structural Changes Related to Melting

Chen, Grace; Stenlund, Arne

doi:10.1128/mcb.22.21.7712-7720.2002

Cited by 26 publications

(20 citation statements)

References 47 publications

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“…To initiate viral replication, E2 binds to DNA in a sequence specific manner at sites flanking the origin of replication, and recruits E1 monomers to the origin. When the first pair of E1 molecules binds to the replication origin, E2 is released from its binding sites through a process of ATP hydrolysis (Chen & Stenlund, 2002). More E1 molecules are recruited and form a double-hexameric complex at the origin that is similar to the eukaryotic Orc-Mcm complex.…”

Section: Discussionmentioning

confidence: 99%

Human Papillomavirus Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein

Thomas

Androphy

2018

J Virol

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iii ACKNOWLEDGEMENTS I would like to thank my supervisor Elliot J. Androphy for his guidance, and encouragement over these many years. I truly appreciate your faith and continuous support.I also want to thank all members of the Androphy lab, for your guidance, critiques, and support.Thank you to my thesis committee, without whom none of this would be possible.Your guidance has been invaluable. Michael Klemsz, Suk-Hee Lee, Lindsey Mayo, and Andy Yu. The Papillomavirus E2 protein is a sequence specific DNA binding protein that recruits cellular factors to its genome in infected epithelial cells. E2 also binds to and loads the viral E1 DNA helicase at the origin of replication. Post-translational modifications of PV E2 have been identified as potential regulators of E2 functions. We recently reported lysine (K) 111 as a target of p300 acetylation in B(bovine)PV that is involved in the regulation of viral transcription. K111 is conserved in most papillomaviruses, so we pursued a mutational approach to query the functional significance of lysine in HPV E2. Amino acid substitutions that prevent acetylation, including arginine, were unable to stimulate transcription and E1 mediated DNA replication. The arginine K111 mutant retained E2 transcriptional repression, nuclear localization, DNA and chromatin binding, and association with E2 binding partners involved in PV transcription and replication. v When directly investigating origin unwinding, the replication defective E2 K111R mutant recruited E1 to the viral replication origin, but surprisingly, unwinding of the duplex DNA did not occur. In contrast, the glutamine K111 mutant increased origin melting and stimulated replication compared to wild type E2. We have identified Topoisomerase I as a key host factor involved in viral replication whose recruitment is dependent on K111 acetylation, and propose a new model for viral origin dynamics during replication initiation. This work reveals a novel activity of E2 necessary for denaturing the viral origin that likely depends on acetylation of highly conserved lysine 111. Human Papillomaviruses and DiseaseThere are over 200 HPVs that have been described and they manifest in a variety of ways; they can be asymptomatic, or they can result in benign lesions (papillomas) or progress to malignancy (de Villiers, Fauquet, Broker, Bernard, & zur Hausen, 2004). In the United States, HPV is the most common sexually transmitted infection and nearly all sexually active adults contract it at least once. E1The PV E1 protein is an essential viral replication factor (Lusky & Botchan, 1985;Ustav, Ustav, Szymanski, & Stenlund, 1991). E1 is the viral helicase and initiates replication by binding to specific sequences on the viral genome with the assistance of the E2 protein (Frattini & Laimins, 1994).There is sequence and functional homology between PV E1 and SV (simian virus) 40 Large T Antigen which allowed for many of the predictions concerning E2 shows differential affinity for the E2BS and it is proposed that at low levels, E2b...

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Section: Discussionmentioning

confidence: 99%

Human Papillomavirus Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein

Thomas

Androphy

2018

J Virol

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“…The DT assembly is dependent on two DNA binding activities. One, in the DNA binding domain (DBD), recognizes four E1 binding sites (E1 BS) in the center of the ori (Chen and Stenlund, 2002; Enemark et al, 2002). The other, located in the helicase domain, includes a β-hairpin structure, and binds non-specifically to the sequences flanking the E1 BS (Schuck and Stenlund, 2005; Liu et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Mechanistic Analysis of Local Ori Melting and Helicase Assembly by the Papillomavirus E1 Protein

Schuck

Stenlund

2011

Molecular Cell

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Summary Preparation of DNA templates for replication requires opening of the duplex to expose single stranded (ss) DNA. The locally melted DNA is required for replicative DNA helicases to initiate unwinding. How local melting is generated in eukaryotic replicons is unknown, but initiator proteins from a handful of eukaryotic viruses can perform this function. Here we dissect the local melting process carried out by the papillomavirus E1 protein. We characterize the melting process kinetically and identify mutations in the E1 helicase and in the ori that arrest the local melting process. We show that a subset of these mutants have specific defects for melting of the center of the ori containing the binding sites for E1 and demonstrate that these mutants fail to untwist the ori DNA. This newfound understanding of how E1 generates local melting suggests possible mechanisms for local melting in other replicons.

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“…First described for the model bovine papillomavirus (BPV) type 1 (4,23,39), the findings have been rapidly extended to mucosal HPV types (2,5,53). The DNA sequence of mucosal HPV origins appears to be well conserved, with an A/T-rich core flanked by E2 binding sites.…”

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Functional Mapping of the Human Papillomavirus Type 16 E1 Cistron

Lace

Anson

Turek

et al. 2008

J Virol

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Replication of the double-stranded, circular human papillomavirus (HPV) genomes requires the viral DNA replicase E1. Here, we report an initial characterization of the E1 cistron of HPV type 16 (HPV-16), the most common oncogenic mucosal HPV type found in cervical and some head and neck cancers. The first step in HPV DNA replication is an initial burst of plasmid viral DNA amplification. Complementation assays between HPV-16 genomes carrying mutations in the early genes confirmed that the expression of E1 was necessary for initial HPV-16 plasmid synthesis. The major early HPV-16 promoter, P97, was dispensable for E1 production in the initial amplification because cis mutations inactivating P97 did not affect the trans complementation of E1؊ mutants. In contrast, E1 expression was abolished by cis mutations in the splice donor site at nucleotide (nt) 226, the splice acceptor site at nt 409, or a TATAA box at nt 7890. The mapping of 5 mRNA ends using rapid amplification of cDNA ends defined a promoter with a transcription start site at HPV-16 nt 14, P14. P14-initiated mRNA levels were low and required intact TATAA (7890). E1 expression required the HPV-16 keratinocyte-dependent enhancer, since cis mutations in its AP-2 and TEF-1 motifs abolished the ability of the mutant genomes to complement E1؊ genomes, and it was further modulated by origin-proximal and -distal binding sites for the viral E2 gene products. We conclude that P14-initiated E1 expression is critical for and limiting in the initial amplification of the HPV-16 genome.High-risk (HR) oncogenic mucosal human papillomavirus (HPV) types are the major cause of most, if not all, carcinomas of the uterine cervix and many other anogenital tumors, and they are found in 20 to 30% of cancers of the head and neck (HNC). HPV type 16 (HPV-16) is the most prevalent HR HPV: it is present in nearly one-half of cervical carcinomas and more than 90% of HPV-associated HNC (reviewed in reference 32). Recently, preventive vaccines against HPV-16 and HPV-18, the second most common HR HPV, have become available. While the vaccines are highly efficacious, they cannot eliminate existing infection. The development of therapies targeted at HPV infection requires a profound understanding of the viral genes involved in replication and their regulation.Upon introduction into the basal layer cell of skin or stratified squamous epithelia, the double-stranded, closed circular HPV plasmid genome undergoes an initial amplification (11,18,45). HPV plasmid amplification in the initial stages of infection appears to be tightly restricted, potentially in part to limit triggering intracellular defense mechanisms by unchecked viral replication and/or to minimize host immune responses to high levels of viral polypeptides. A low steady-state viral copy number in established HPV infections suggests limited levels of cellular factors or the regulated synthesis of viral factors responsible for supporting HPV replication.Papillomaviral DNA replication requires an origin of replication (ori) and two...

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Sequential and Ordered Assembly of E1 Initiator Complexes on the Papillomavirus Origin of DNA Replication Generates Progressive Structural Changes Related to Melting

Cited by 26 publications

References 47 publications

Human Papillomavirus Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein

Human Papillomavirus Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein

Mechanistic Analysis of Local Ori Melting and Helicase Assembly by the Papillomavirus E1 Protein

Functional Mapping of the Human Papillomavirus Type 16 E1 Cistron

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